This application addresses broad Challenge Area (06) Enabling Technologies and Specific Challenge Topic 06-HG-102: Technologies for obtaining genomic, proteomic, and metabolomic data from individual viable cells in complex tissues. Perhaps the greatest challenge in the area of proteomics is to develop methods that can report on the abundances and post-translational modification states of many different proteins in a single cell or cell type obtained with high spatial and temporal resolution from complex, living tissue. There are two fundamental issues that need to be addressed in order to meet this challenge. First, new and innovative sample collection methods are needed to enable the fast and efficient recovery of material from single cells embedded in live tissue. Second, highly sensitive analytical techniques are needed that can accurately quantify proteins in a multiplexed fashion in extremely small sample sizes (1-100 cells). Here, a new technology - termed "microlysis technology" - is described that enables the collection of lysates from single cells embedded in complex, living tissue. This technology uses mobile laminar flow of lysis buffer to efficiently lyse a single cell in approximately three to five seconds and to recover the lysate in a volume of approximately two nanoliters. First, a strategy is presented to build an instrument that automates this technology. Second, a plan is presented to couple this technology with lysate microarray technology in order to quantify protein abundances and post-translational modification states in a highly multiplexed and high-throughput fashion. These development efforts will be focused on the most challenging of tissues, the mammalian brain, which comprises thousands of distinct cell types and hence has defied most biochemical and proteomics efforts to characterize it at the molecular level. The technology presented here can realistically be developed in a two-year timeframe. If this work is successful, a new company will be launched at the end of the funding period to commercialize microlysis technology. Funding of this proposal will stimulate the economy through the immediate acquisition of instrumentation, through the hiring of two postdoctoral fellows, and through the founding of a new company, thereby creating additional jobs. On a scientific level, the ability to collect lysates from single cells embedded in complex living tissue will have a profound effect on the fields of genomics, proteomics, and metabolomics. To date, efforts in these areas have relied either on cultured cells, which have questionable physiological relevance, or on whole tissue lysates, which comprise dozens to hundreds of distinct cell types. Microlysis technology will enable the physiologically relevant study of biomolecules in virtually any solid tissue. One of the greatest challenges in biochemistry is to study dozens to hundreds of molecules simultaneously using single cells or in single cell types obtained from complex, living tissue. Here, we propose a new technology, termed "microlysis technology", which enables the automated collection and quantitative analysis of single cells embedded in acute brain slices. This technology will have a profound impact on the fields of genomics, proteomics, and metabolomics since it will enable researchers to study biomolecules in a physiologically relevant fashion in virtually any solid tissue.